Antiviral drugs prolong survival in murine recessive dystrophic epidermolysis bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare inherited skin disease characterized by defects in type VII collagen leading to a range of fibrotic pathologies resulting from skin fragility, aberrant wound healing, and altered dermal fibroblast physiology. Using a novel in vitro model of fibrosis based on endogenously produced extracellular matrix, we screened an FDA-approved compound library and identified antivirals as a class of drug not previously associated with anti-fibrotic action. Preclinical validation of our lead hit, daclatasvir, in a mouse model of RDEB demonstrated significant improvement in fibrosis as well as overall quality of life with increased survival, weight gain and activity, and a decrease in pruritus-induced hair loss. Immunohistochemical assessment of daclatasvir-treated RDEB mouse skin showed a reduction in fibrotic markers, which was supported by in vitro data demonstrating TGFβ pathway targeting and a reduction of total collagen retained in the extracellular matrix. Our data support the clinical development of antivirals for the treatment of patients with RDEB and potentially other fibrotic diseases.

24th Oct 2023 1st Editorial Decision 24th Oct 2023 Dear Dr. South, Thank you for the submission of your manuscript to EMBO Molecular Medicine.We have now received feedback from the three reviewers who agreed to evaluate your manuscript.All three referees recognize potential interest of the study but also raise important criticism that should be addressed in a major revision.If you would like to discuss further the points raised by the referees, I am available to do so via email or video.Let me know if you are interested in this option.
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Zeljko Durdevic Editor EMBO Molecular Medicine ***** When submitting your revised manuscript, please carefully review the instructions that follow below.We perform an initial quality control of all revised manuscripts before re-review; failure to include requested items will delay the evaluation of your revision.
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EMBO Molecular Medicine has a "scooping protection" policy, whereby similar findings that are published by others during review or revision are not a criterion for rejection.Should you decide to submit a revised version, I do ask that you get in touch after three months if you have not completed it, to update us on the status.
Please note: When submitting your revision you will be prompted to enter your funding and payment information.This will allow Wiley to send you a quote for the article processing charge (APC) in case of acceptance.This quote takes into account any reduction or fee waivers that you may be eligible for.Authors do not need to pay any fees before their manuscript is accepted and transferred to the publisher.Dystrophic epidermolysis bullosa (DEB) is a devastating genetic disorder associated with fibrotic scarring.Tartaglia et al. used a novel in vitro model based on endogenously produced extracellular matrix to screen an FDA-approved compound library and identified antivirals as a drug class not yet known for anti-fibrotic action.Their lead drug candidate, daclatasvir, was then evaluated in a mouse model of DEB, where it increased survival of affected animals and mitigated the disease phenotype.Daclatasvir had been approved for clinical use in combination with other antiviral agents as therapy of chronic hepatitis C, but was withdrawn by the sponsor in 2019.However, Tartaglia et al. also screened a focused 240 antiviral drug library and report 57 hits across two DEB dermal fibroblast populations that delayed matrix detachment.Overall, the paper presents a solid set of data, adding something new and relevant to the field of epidermolysis bullosa research.The results support clinical development of antivirals not only for the treatment of DEB but may also pave the way to tackle the burden of other fibrotic diseases.

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The following comments need to be addressed to improve the manuscript: 1. What does Supplemental Fig. 2 show?The PDF lacks a legend with relevant information, e.g., which controls were used.
2. The authors describe the effects of numerous compounds on DEB fibroblasts.That disease phenotype serves as control throughout the paper.It would also be interesting to know what happens if selected compounds are applied to wild-type dermal fibroblasts, and the respective data for two compounds are actually reported in Supplemental Fig. 4. Have the authors looked at this more systematically?Any effect that is seen also on wild-type fibroblasts would be more than a rescue effect! 3. Why did the authors investigate only individual mRNAs by qPCR?Results of a complete array would have provided relevant additional information.
4. Clarity for the nonspecialist reader could be improved, especially in the results section.

Referee #2 (Comments on Novelty/Model System for Author):
There is a huge unmet need to treat fibrosis in RDEB.They included a diverse range of RDEB patient and mouse samples.They tested a large range of molecules with the assay.

Referee #2 (Remarks for Author):
This study developed assays to assess fibrosis and TGFB in RDEB Please explain any relation between elevated basic FGF found in RDEB and the TGFB and fibrosis Do these drugs that reduce fibrosis impact basic FGF as this also stimulates fibrosis?How was quality of life measured in these mice?Referee #3 (Remarks for Author): In the manuscript "Antiviral drugs prolong survival in murine recessive dystrophic epidermolysis bullosa" Tartaglia and colleagues describe a screen of FDA approved drugs to identify compounds to be used in RDEB, a skin fragility disorder characterized by high level of fibrosis.As readout they use an in vitro system i.e., the detachment of cell matrices from culture plates.Anti-viral drugs have a positive effect in vitro, reduce fibrosis characteristics, and two lead compounds are further characterized.Finally, the best scoring compound daclatasvir is tested in a preclinical mouse model and has positive effects on fibrosis, life quality and span.This is an interesting and well written manuscript that follows a trend observable in the last years: the repurposing of drugs to treat rare diseases for which no causal therapies are available.The findings are interesting and promise a swift translation, addressing an important and unmet clinical need.With respect to the presented data several questions remain which should be addressed prior publication.

Major concerns:
(1) The screen data should be made publicly available in form of a supplemental excel spreadsheet.How were selections made?Right now, one cannot recapitulate how drugs were selected and which quantitative measures/cutoffs and/or statistics were used to define targets.This is absolutely critical so that readers can perform their own statistical analyses (which may yield different shortlists).
(2) I cannot follow the logic of hydroxyproline measurements (Figure 1I and 4F).What is the major conclusion of these experiments and how does this relate to literature knowledge?When there is more hydroxyproline in the medium of non-EB cells does it mean that there is more secreted/soluble collagen?This is contradicting the fibrosis phenotype.Authors should perform additional tests e.g., western blots or proteomics to directly address collagen levels in medium.Hydroxyproline is just a proxy and may be present in other proteins or in a free form.It could also indicate a difference in collagen crosslinking and reflect hydroxyproline accessibility (more than collagen amount as misleadingly indicated by the axis labels).Also, axis labels should be corrected and indicate the actual measurement.
(3) Used drug concentrations in screen: how do the 10 M relate to clinical relevant concentrations?Are drugs prescribed in a similar range, are serum levels in a similar range?(4) Figure 4D: data spread of controls should be considered.It appears that control values have been set/normalized to 1.If this is done before significance testing, most changes might wrongly appear significant.

Minor points:
(1) In Figure 4A, 1 M of daclatasvir seems to have not a significant effect, in contrast to Figure 4B.How are these two figure panels related?How is the difference explained?(2) In the discussion a Figure 4H with AFM data is mentioned which is not included in the manuscript.This should be corrected.It would be interesting to compare AFM measurements of EB and non-EB matrices.

Referee #1 (Remarks for Author):
Dystrophic epidermolysis bullosa (DEB) is a devastating genetic disorder associated with fibrotic scarring.Tartaglia et al. used a novel in vitro model based on endogenously produced extracellular matrix to screen an FDA-approved compound library and identified antivirals as a drug class not yet known for anti-fibrotic action.Their lead drug candidate, daclatasvir, was then evaluated in a mouse model of DEB, where it increased survival of affected animals and mitigated the disease phenotype.Daclatasvir had been approved for clinical use in combination with other antiviral agents as therapy of chronic hepatitis C, but was withdrawn by the sponsor in 2019.However, Tartaglia et al. also screened a focused 240 antiviral drug library and report 57 hits across two DEB dermal fibroblast populations that delayed matrix detachment.Overall, the paper presents a solid set of data, adding something new and relevant to the field of epidermolysis bullosa research.The results support clinical development of antivirals not only for the treatment of DEB but may also pave the way to tackle the burden of other fibrotic diseases.
Reply: We thank the Reviewer for their thorough assessment of our manuscript and we are very happy that the Reviewer appreciates the novelty and clinical potential of our findings.We appreciate the Reviewer's recommendations which we have addressed as detailed below.We believe the additions and modifications suggested by the Reviewer have greatly improved our manuscript.
The following comments need to be addressed to improve the manuscript: 1.What does Supplemental Fig. 2 show?The PDF lacks a legend with relevant information, e.g., which controls were used.
Reply: We apologize for the oversight regarding the legend for Supplemental Figure 2 which we agree was not adequately describing the data presented.We have replaced the legend for Supplemental Figure 2 to clearly describe the data we are presenting and we thank the Reviewer for flagging this error.

The authors describe the effects of numerous compounds on DEB fibroblasts. That disease phenotype serves as control throughout the paper. It would also be interesting to know what happens if selected
compounds are applied to wild-type dermal fibroblasts, and the respective data for two compounds are actually reported in Supplemental Fig. 4. Have the authors looked at this more systematically?Any effect that is seen also on wild-type fibroblasts would be more than a rescue effect!Reply: We agree with the Reviewer that it is important to determine whether antivirals represent an active process in the absence of a disease phenotype.As the Reviewer pointed out we had looked at our lead hit compounds in normal, wild-type, fibroblasts in Supplemental Figure 4. We have now added Western blotting data to confirm that our lead antiviral hit compounds do not affect Collagen I, phosphorylated SMAD3 or Akt at the protein level (new Supplemental Figure 4).

Why did the authors investigate only individual mRNAs by qPCR? Results of a complete array would have provided relevant additional information.
Reply: We thank the reviewer for the suggestion of running a complete array to look at the effects of daclatasvir and idoxuridine on RDEB fibroblasts.In response, and in addition to our focused qPCR approach to assess collagen processing enzymes we have added RNA sequencing data comparing daclatasvir with vehicle control in RDEB fibroblasts.These new data agree with our observations that 26th Jan 2024 1st Authors' Response to Reviewers daclatasvir reduces collagen I mRNA and has little effect on the collagen processing enzyme genes we originally presented in the old Supplemental Figure 5.We have added the RNA sequencing data to the new Supplemental Figure 5.

Clarity for the nonspecialist reader could be improved, especially in the results section.
We have reviewed the results section and altered some of the text as indicated with a view to improve readability and explanation of our approach for the non-specialist reader.We hope these improvements adequately address this important comment.

Referee #2 (Comments on Novelty/Model System for Author):
There is a huge unmet need to treat fibrosis in RDEB.They included a diverse range of RDEB patient and mouse samples.They tested a large range of molecules with the assay.
Reply: We thank the Reviewer for their assessment of our manuscript and their appreciation of the range of compounds and breadth of samples used in our study.

Referee #2 (Remarks for Author): This study developed assays to assess fibrosis and TGFB in RDEB Please explain any relation between elevated basic FGF found in RDEB and the TGFB and fibrosis
Reply: The Reviewer makes a very good point since FGF has been linked with fibrosis in non-RDEB settings and there is a study from the late 90's identifying increased FGF in urine of RDEB patients (Arbiser et al, 1998).Previously reported unbiased screens looking at gene expression or protein changes in RDEB fibroblasts compared to non-RDEB fibroblasts have not identified altered levels of FGF (Küttner et al, 2014;Küttner et al, 2013;Ng et al, 2012;Nyström et al, 2013) and this is the reason we had not previously looked at levels in our assays.These dermal fibroblast specific data would indicate a separate source for FGF in RDEB patients, perhaps muscle, connective tissues or the kidney itself since these tissues are reported to have relatively high levels of FG2 when reviewing the Protein Atlas (https://www.proteinatlas.org/ENSG00000138685-FGF2/tissue).However, the Reviewer makes an excellent suggestion and in response we have performed Western blotting to compare protein levels of FGF in RDEB and non-EB fibroblasts (Figure R1).These experiments found no significant change in fibroblast FGF2 protein levels comparing RDEB and non-EB; however, SB treatment significantly reduced FGF2 expression in EB while TGFβ increased FGF2 expression in non-EB, although this was not significant in the three populations of non-EB cells we analyzed.These data are in line with published studies identifying a relationship between TGF and FGF (Strand et al, 2014) but do not identify differences between RDEB and non-EB.

Do these drugs that reduce fibrosis impact basic FGF as this also stimulates fibrosis?
Reply: We thank the reviewer for their suggestion to look at the impact of antiviral treatment on FGF levels in RDEB fibroblasts.Western blotting experiments found no significant effect on FGF for our two lead hit antiviral compounds as shown (Figure R2).

How was quality of life measured in these mice?
Reply: Quality of life for the RDEB mice was measured using activity (more energy and less pain to move aroundsee Figure 5B), hair retention (a sign of diminished pruritussee Figure 5D), and weight gain (increased appetitesee Figure 5C).

Referee #3 (Remarks for Author):
In the manuscript "Antiviral drugs prolong survival in murine recessive dystrophic epidermolysis bullosa" Tartaglia and colleagues describe a screen of FDA approved drugs to identify compounds to be used in RDEB, a skin fragility disorder characterized by high level of fibrosis.As readout they use an in vitro system i.e., the detachment of cell matrices from culture plates.Anti-viral drugs have a positive effect in vitro, reduce fibrosis characteristics, and two lead compounds are further characterized.Finally, the best scoring compound daclatasvir is tested in a preclinical mouse model and has positive effects on fibrosis, life quality and span.This is an interesting and well written manuscript that follows a trend observable in the last years: the repurposing of drugs to treat rare diseases for which no causal therapies are available.The findings are interesting and promise a swift translation, addressing an important and unmet clinical need.
Reply: We thank the Reviewer for their astute and thorough review of our manuscript and we greatly appreciate the questions, comments and suggestions which we have endeavored to address below.We are happy that the Reviewer believes our study to be well written, interesting and have clinical value.
With respect to the presented data several questions remain which should be addressed prior publication.Major concerns: (1) The screen data should be made publicly available in form of a supplemental excel spreadsheet.How were selections made?Right now, one cannot recapitulate how drugs were selected and which quantitative measures/cutoffs and/or statistics were used to define targets.This is absolutely critical so that readers can perform their own statistical analyses (which may yield different shortlists).
Reply: We thank the reviewer for their interest in our screen data and have provided a supplemental excel table as part of the SourceData for Figure 3, as required by the Journal.We have also added more details about our quantitative measures in the methods section in terms of how our selections were made.Essentially, the initial screen was performed in triplicate and the average number of days for detachment of the matrix was used to compare vehicle control.Those compounds that delayed detachment longer than 2 days after control were taken forward in the secondary screen.The screens were observational in nature and did not include statistics to define targets.We have added this detail to the manuscript.
(2) I cannot follow the logic of hydroxyproline measurements (Figure 1I and 4F).What is the major conclusion of these experiments and how does this relate to literature knowledge?When there is more hydroxyproline in the medium of non-EB cells does it mean that there is more secreted/soluble collagen?This is contradicting the fibrosis phenotype.Authors should perform additional tests e.g., western blots or proteomics to directly address collagen levels in medium.Hydroxyproline is just a proxy and may be present in other proteins or in a free form.It could also indicate a difference in collagen crosslinking and reflect hydroxyproline accessibility (more than collagen amount as misleadingly indicated by the axis labels).Also, axis labels should be corrected and indicate the actual measurement.
Reply: We appreciate the reviewers' questions regarding the hydroxyproline assay and we now attempt to provide further clarification in the manuscript.Our original major conclusions from the assay experiments were that 1) RDEB matrices retain a greater proportion of their collagen compared with non-EB matrices, in line with fibrosis in RDEB patients and accelerated detachment of RDEB matrices, 2) Stimulating or inhibiting TGFβ in non-EB or RDEB matrices (respectively) reverses the matrix retention trends, and 3) while the antivirals promoted the non-EB collagen retention phenotype in RDEB matrices, Idoxuridine stimulated more collagen retention in non-EB matrices and that contributed to our decision not to pursue Idoxuridine to in vivo trials.We agree and now acknowledge in the revised manuscript that hydroxyproline is indeed a surrogate for collagen since other proteins are hydroxylated at their proline residues (particularly proteins secreted by dermal fibroblasts (Küttner et al., 2013).We also acknowledge that the measurements we present are not absolute values since the insolubility of collagen presents challenges for absolute values in the matrices and the stability of collagens and hydroxylated proline residues in the media over the seven days of the experiment will likely be influenced by multiple factors.We have added clarity to the manuscript to address these issues and we have altered the graph axis to reflect the actual measurements as requested.Collagen I levels in the matrix are presented throughout the manuscript and the consistent increase as a measure of fibrosis in our study is the level of collagen I in the matrix.Collagen secreted into the media is soluble and not processed into fibrils or other secondary structures and is subject to degradation by cells in culture and is not the focus of our study since we have used cells own ECM (matrix) detachment from tissue culture to identify antiviral drugs.We have previously published that although the level of collagen I is increased in RDEB, the secretion (measured as a ratio of intracellular to extracellular collagen) is unaltered (Cao et al, 2022).Other collagens do show changes in their secretion and intracellular retention, such as collagen 12, but how this relates to matrix composition and detachment is unknown.We feel that a proteomics study of secreted vs ECM retained collagen is beyond the scope of our study and we refer the Reviewer to the excellent work of Jorn Dengjel, Victoria Kuttner and colleagues who report on beautiful proteomics in RDEB and normal fibroblasts (Küttner et al., 2014;Tölle & Dengjel, 2019).
(3) Used drug concentrations in screen: how do the 10 µM relate to clinical relevant concentrations?Are drugs prescribed in a similar range, are serum levels in a similar range?
We appreciate the Reviewer's point and agree that 10µM cannot be considered a clinically relevant concentration for all compounds since this will vary from compound to compound.For the purpose of a screen we started with 10µM and moved forward with our lead hits at 1µM.Our rationale for starting at a relatively high concentration was to avoid taking compounds that could potentially be toxic in the setting of RDEB since patients with RDEB suffer from failure to thrive and often are at weights associated with children, which would lead to increased drug exposure if taking a standard dose of an approved therapy.We wanted to make sure that we didn't pursue drugs with potential side effects in an already challenging clinical setting.For the animal work we do use a physiologically and clinical relevant dose in line with the dose used clinically for daclatasvir (Zakaria & El-Sisi, 2020).
(4) Figure 4D: data spread of controls should be considered.It appears that control values have been set/normalized to 1.If this is done before significance testing, most changes might wrongly appear significant.
Reply: We appreciate the Reviewer's comment that indeed normalizing controls to 1 has the potential to influence significance for certain datasets.In this instance we are analyzing the change in protein abundance comparing treatment with vehicle control using multiple patient primary populations of fibroblasts with Western blotting.Given that there is considerable variation in both baseline signal from separate populations as well as intensities of chemiluminescence, which itself does not always provide consistent values comparing different experiments, the appropriate way to measure change in protein abundance after treatment is to normalize the data to vehicle control.We consulted our local biostatistician, Dr. Tingting Zhan who confirmed that this approach is the correct practice for this type of analysis.Our raw data is available for review in the SourceData associated with each figure as per journal policy.

Minor points:
(1) In Figure 4A, 1 µM of daclatasvir seems to have not a significant effect, in contrast to Figure 4B.How are these two figure panels related?How is the difference explained?
Reply: We thank the reviewer for pointing out this discrepancy which was a result of completely separate experiments performed at very different times.The original experiments shown in the old Figure 4A were used to determine the best dose moving forward from the screen.1uM was identified and taken forward, and the old Figure 4B confirmed that this dose indeed consistently delayed detachment in 6 separate RDEB populations, comparing to vehicle alone control each time.Whilst daclatasvir consistently delayed detachment in the old Figure 4A the data were under powered to reach significance.As a result, we have moved the old Figure 4A to the new Supplementary Figure 3.The new Figure 4 now begins with the original separate, and suitably powered experiments using 6 different RDEB populations showing that our lead screen hits delay detachment in our in vitro assay of fibrosis.
(2) In the discussion a Figure 4H with AFM data is mentioned which is not included in the manuscript.This should be corrected.It would be interesting to compare AFM measurements of EB and non-EB matrices.
Reply: We thank the reviewer for catching the typo and have corrected the discussion to point out Figure 4G (new 4F).We have also shown AFM measurements of RDEB and non-EB matrices in Figure 1F.
9th Feb 2024 1st Revision -Editorial Decision 9th Feb 2024 Dear Dr. South, Thank you for the submission of your revised manuscript to EMBO Molecular Medicine.I am pleased to inform you that we will be able to accept your manuscript pending the following final amendments: 1) Figures: -Remove all figures from the main manuscript file and leave only their legends placed after "References".All supplementary figures should be moved to Appendix file and renamed to Appendix Figure S1 etc.For more information on figure presentation please check "Author Guidelines".https://www.embopress.org/page/journal/17574684/authorguide#datapresentationformat-We notice that Western blot images are of low resolution.Please display images in all figures in higher resolution.
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Referee #3 (Remarks for Author):
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EMBO Press Author Checklist USEFUL LINKS FOR COMPLETING THIS FORM
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Abridged guidelines for figures 1. Data
The data shown in figures should satisfy the following conditions: New materials and reagents need to be available; do any restrictions apply?Not Applicable

Antibodies
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(Reagents and Tools For antibodies provide the following information: -Commercial antibodies: RRID (if possible) or supplier name, catalogue number and or/clone number -Non-commercial: RRID or citation

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Cell materials
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(Reagents and Tools Cell lines: Provide species information, strain.Provide accession number in repository OR supplier name, catalog number, clone number, and/OR RRID.

Not Applicable
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Experimental animals
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(Reagents and Tools

Plants and microbes
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Reporting
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Yes
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Not Applicable
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Not Applicable
If publicly available data were reused, provide the respective data citations in the reference list.

Not Applicable
The MDAR framework recommends adoption of discipline-specific guidelines, established and endorsed through community initiatives.Journals have their own policy about requiring specific guidelines and recommendations to complement MDAR.

Figure R1 .
Figure R1.TGFβ regulation impacts FGF2 expression but shows no difference between RDEB and non-EB.(Left) Western blots of FGF2 and GAPDH in RDEB (n=4) and non-EB (n=3) fibroblasts with SB or TGFβ treatment.(Middle) Quantification of blot presented as graph showing mean ± SEM of FGF2 expression relative to GAPDH.Ordinary one-way ANOVA with Šídák test for significance.**p<0.01;ns, not significant.(Right) Quantification of blot presented as graph showing mean ± SEM of FGF2 expression relative to GAPDH.Unpaired t-test for significance.

Figure R2 .
Figure R2.Antivirals do not affect FGF2 expression in RDEB fibroblasts.(Left) Western blots of FGF2 and GAPDH in RDEB fibroblasts (n=4) with vehicle control, idoxuridine, or daclatasvir treatment.(Right) Quantification of blot presented as graph showing mean ± SEM of FGF2 expression relative to GAPDH.RN one-way ANOVA for significance.
11) Please provide a point-by-point letter INCLUDING my comments as well as the reviewer's reports and your detailed responses (as Word file).I look forward to reading a new revised version of your manuscript as soon as possible.Instructions to submit your revised manuscript *** *** PLEASE NOTE *** As part of the EMBO Publications transparent editorial process initiative (see our Editorial at https://www.embopress.org/doi/pdf/10.1002/emmm.201000094),EMBO Molecular Medicine will publish online a Review Process File to accompany accepted manuscripts.
Figures are not edited by the production team.All lettering should be the same size and style; figure panels should be indicated by capital letters (A, B, C etc).Gridlines are not allowed except for log plots.Figures should be numbered in the order of their appearance in the text with Arabic numerals.Each Figure must have a separate legend and a caption is needed for each panel.*Additional important information regarding figures and illustrations can be found at https://bit.ly/EMBOPressFigurePreparationGuideline.See also figure legend preparation guidelines: https://www.embopress.org/page/journal/17574684/authorguide#figureformat

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definitions of statistical methods and measures: (Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section) Table, Materials and Methods, Figures, Data Availability Section) Table, Materials and Methods, Figures, Data Availability Section) Table, Materials and Methods, Figures, Data Availability Section) Table, Materials and Methods, Figures, Data Availability Section) Provide species, strain, sex, age, genetic modification status.Provide accession number in repository OR supplier name, catalog number, clone number, OR RRID.

In which section is the information available?
Table, Materials and Methods, Figures, Data Availability Section) (Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section) If collected and within the bounds of privacy constraints report on age, sex and gender or ethnicity for all study participants.

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Please complete ALL of the questions below. Select "Not Applicable" only when the requested information is not relevant for your study. if
n<5, the individual data points from each experiment should be plotted.Any statistical test employed should be justified.Source Data should be included to report the data underlying figures according to the guidelines set out in the authorship guidelines on Data Each figure caption should contain the following information, for each panel where they are relevant: a specification of the experimental system investigated (eg cell line, species name).theassay(s)and method(s) used to carry out the reported observations and measurements.anexplicitmention of the biological and chemical entity(ies) that are being measured.anexplicitmention of the biological and chemical entity(ies) that are altered/varied/perturbed in a controlled manner.ideally,figurepanels should include only measurements that are directly comparable to each other and obtained with the same assay.plotsinclude clearly labeled error bars for independent experiments and sample sizes.Unless justified, error bars should not be shown for technical the exact sample size (n) for each experimental group/condition, given as a number, not a range; a description of the sample collection allowing the reader to understand whether the samples represent technical or biological replicates (including how many animals, litters, cultures, etc.).a statement of how many times the experiment shown was independently replicated in the laboratory.This checklist is adapted from Materials Design Analysis Reporting (MDAR) Checklist for Authors.MDAR establishes a minimum set of requirements in transparent reporting in the life sciences (see Statement of Task: 10.31222/osf.io/9sm4x).Please follow the journal's guidelines in preparing your the data were obtained and processed according to the field's best practice and are presented to reflect the results of the experiments in an accurate and unbiased manner.

Checklist for Life Science Articles (updated January Study protocol Information included in the manuscript? In which section is the information available?
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In which section is the information available?
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Sample definition and in-laboratory replication Information included in the manuscript? In which section is the information available?
(Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section)In the figure legends: state number of times the experiment was replicated in laboratory.

Ethics Ethics Information included in the manuscript? In which section is the information available?
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Use Research of Concern (DURC) Information included in the manuscript? In which section is the information available?
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granting approval and reference number for
the regulatory approval provided in the manuscript?

and III randomized controlled trials
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